Throughout this application, various publications are referred to by Arabic numerals within parenthesis. Full citations for these references may be found at the end of the specification, immediately preceding the claims. The disclosures of these publications are hereby incorporated by reference in order to more fully describe the state of the art at the time of the invention described and claimed herein.
The present invention is based on the surprising discovery that undenatured whey protein concentrate has an enhanced immunological effect. More specifically, the invention relates to the effect of the oral administration of whey protein concentrate in undenatured conformation on the immune response to sheep red blood cells, host resistance to pneumococcal infections, development of chemically induced colon carcinoma and tissue glutathione.
The present invention shows the correlation between the undenatured conformation of whey protein concentrate (w.p.c.) and host immunoenhancement whereby chemical indices of denaturation are given and the demonstration that the same crucial role of molecular conformation (undenatured state) applies to glutathione GSH promotion, which is the other major biological activity of w.p.c. Equally important is the demonstration that another protein source such as egg white, with the same high cysteine content as w.p.c. does not enhance GSH synthesis, further demonstrating the specificity of w.p.c. with respect to the described biological activity.
Whey and whey protein have been utilized from time immemorable for nutritional purposes. In addition, whey was recommended in folk and ancient medicine for the treatment of various diseases .sup.(1,2) and, in one instance, lifetime feeding of hamsters with a whey protein diet has been shown to promote longevity with no explanation given.sup.(3,4).
Dairy products are widely used as a good source of nutrition. In addition, claims have been made to the effect that fermented whole milk (yogurt) is beneficial in the management of some types of intestinal infections. Certain dietary regimes based on ill defined natural or cultured dairy products are said to be associated with long life expectancy in some regions of the U.S.S.R., for example, Georgia.
Since time immemorial, serum lactis, which is latin for milk serum or whey, has been administered to the sick for the treatment of numerous ailments. In 1603, Baricelli reported on the therapeutic use of cow or goat milk serum sometimes mixed with honey or herbs. The spectrum of illnesses treated with whey include jaundice, infected lesions of skin, those of the genito-urinary tract with purulent secretions, gonorrhea, epilepsy, quartan fever and other febrile states of different origins. Indeed, the common denominator of most of these illnesses appears to be a septic condition. Although physicians of both ancient times and of the middle ages agreed that whey treatment should be carried out over a period of several days, a difference of opinion appear to exist concerning the daily amount prescribed. Thus, Galen, Hippocrates and Dioscoride insisted on a minimum daily amount of two 12 ounce latin libras, and up to five libras a day according to gastric tolerance. This would represent between one to two liters of whey a day. Baricelli on the other hand, reflecting the trend of his time, limited the amount prescribed to one libra a day, given in fractionated doses on an empty stomach.
Since then, numerous articles published in Europe through the 17th, 18th and 19th centuries have advocated the therapeutic use of whey. In an Italian textbook published in the middle of the 19th century.sup.(15), at the dawn of scientific medicine, an interesting distinction is made between whole milk and milk serum. Milk is recommended firstly as a nutrient especially in patients with strictures of the gastro intestinal track. In this respect the author emphasises that the benefits of the then popular "milk therapy" of cachexia and tuberculosis are due only to the nutritional property of milk. Secondly, the milk was prescribed in the treatment of poisoning because milk components would presumably neutralize ingested toxic material. Thirdly, milk therapy was suggested for the alleged capacity of this fluid to coat and soothe ulcers of the gastrointestinal track. Milk serum, on the other hand, was advocated in the treatment of pneumonitis, acute inflammatory diseases of the intestines and urogenital track, in spite of its recognized lower nutritional quality. Finally, the author emphasized the ineffectiveness of whey in the treatment of disorders of the nervous system.
The prime difference between whey (serum lactis) and whole milk is the near absence in the former of the caseins, the casein-bound calcium and phosphate, most of the fat and the fat soluble vitamins. The actual concentration in whey of "whey proteins" is usually similar to that in milk. Hence quantitative differences between whey and milk could not be construed to represent a key factor in the alleged therapeutic effect of whey treatment because, if any, they imply the lack, in whey, of some important nutrients. Some previously collected data .sup.(5-10) of the present inventors provide a scientific background to the presumed benefit of intensive treatment with "serum lactis". The importance of the characteristic amino acid and peptide profile of whey protein concentrate in the immune enhancing effect of the whey protein concentrate (WPC) has been shown. The caseins represent 80% of the total protein content of cows milk while WPC is only 20%. Hence, it is conceivable that it is the separation of WPC from the caseins in whey which represents the crucial qualitative change, since this would render the amino acid profile and associated small peptides patterns of whey proteins unaltered by that of the caseins, once the digestive process has released free amino acids from all ingested proteins.
The search for the possible mechanism of immunoenhancement by whey protein feeding has revealed to us the provocative possibility that whey protein concentrate may contribute to a broader biological effect of a protective nature involving susceptibility to cancer and general detoxification of environmental agents. All these conditions appear to be somehow related to changes in glutathione which is a ubiquitous element exerting a protective effect against superoxide radicals and other toxic agents.
Glutathione is a tripeptide thiol (L-gamma-glutamyl-L-cysteinylglycine) with a broad range of vital functions that include detoxification of xenobiotics and protection of cells against oxygen intermediates and free radicals, by-products of oxygen-requiring metabolism.sup.(42-45). Modulation of intracellular glutathione affects the proliferative immune response of lymphocytes which may be inhibited by oxidative injury.sup.(46-48). Glutathione protect the cells against radiation and alkylating agents.sup.(49-50). Age-related or experimentally induced glutathione depletion in the lens is associated with cataract formation.sup.(51,52). Oxidative DNA damage is rapidly and effectively repaired. The human body is continually repairing oxidized DNA. A small fraction of unrepaired lesions, however, could cause permanent changes in DNA and might be a major contributor to old age diseases and cancer.sup.(53). Indeed, several age associated diseases may be induced by free radicals.sup.(54). It appears that whereas data on age-related changes in tissue vitamin E and other antioxidants are, at best, contradictory.sup.(55), the tissue glutathione levels are more consistently reported to decline with old age in laboratory animals.sup.(56,57) and man.sup.(58-61).
For these reasons there has been interest in the factors that influence intracellular glutathione synthesis and especially in ways of increasing cellular levels of glutathione.
Glutathione is composed of three amino acids: glutamic acid, glycine and cysteine. Availability of cysteine is a limiting factor in the synthesis of glutathione.sup.(62,63). Cysteine is derived from dietary protein and by trans-sulfuration from methionine in the liver. Various methods have been tried in order to increase cellular levels of glutathione. Administration of free cysteine is not an ideal method because this amino acid is rapidly oxidized, toxio.sup.(64) and may actually cause glutathione depletion.sup.(65). Similar problems have been encountered with i.p. injection of N-acetyl cysteine to rats, although oral administration of this compound appeared to prevent paracetamol-induced glutathione depletion.sup.(65). Administration of compounds that are transported and converted intracellularly into cysteine, such as L-2-oxothiazolidine-4-carboxylate are useful in increasing cellular glutathione(66) acting as an intracellular delivery system for cysteine. Hepatic glutathione doubled four hours after injection, returned to normal 8 hours later but was below normal after 16 hours.sup.(66). Another approach for increasing tissue glutathione levels was found in s.c. injection of gamma glutamylcyst(e)ine in mice: glutathione increased in the kidney by about 55%, 40-60 minutes after injection, returning to near control values 2 hours later.sup.(67). The administered compound is transported intact and serves as a substrate for glutathione synthetase. It was also reported that about 2 hours after i.p. administration of gamma-glutamyl cysteinyl-glycyl monomethyl (or monoethyl) ester to mice, the liver and kidney glutathione levels were doubled, with return to normal values after 8 hours.sup.(68). Similar increases in glutathione tissue levels were attained by Meister by administering an alkyl monoester of glutathione (U.S. Pat. No. 4,784,685, Nov. 15th, 1988), to mice. Such esters are transported into tissue cells, and are deesterified within the cells, thus leading to increased cellular levels of glutathione. The kinetics of tissue glutathione increments attained with this method are similar to those described following i.p. injection of methyl or ethyl esters of glutathione.sup.(68). The effectiveness of these methods has been clearly demonstrated in acute experiments.sup.(68,69). (U.S. Pat. No. 4,784,685); in mice treated with L-2-oxothiazolidine-4-carboxylate the expected drop in glutathione tissue level subsequent to acetaminophen injection, was replaced by an actual increase in tissue glutathione values and survival. Other methods to increase tissue glutathione levels are based on the "overshoot" of glutathione concentration, following depletion by diethylmaleate or BSO. These studies were done in vitro on murine cell lines.sup.(70). Also pre-exposure of rats to hypoxia was found to increase lung glutathione.sup.(71).
The administration of glutathione itself is of little consequence on tissue glutathione levels, because it apparently cannot be transported intact across the cell membrane.sup.(68).
Some of said methods of increasing intracellular levels of glutathione concentration are either toxic or dangerous owing to the risks related to the initial phase of glutathione depletion. The methods involving the use of gamma-glutamyloyst(e)ine, athiazolidine or glutathione esters (U.S. Pat. No. 4,784,685) offer an interesting possibility for short term intervention. However, their long term effectiveness in producing sustained elevation of cellular glutathione has not been shown, nor has the possible toxicity of their long term use been disproved. Indeed, glutathione and glutathione disulfide were found to be positive in the most commonly used short term tests for carcinogenicity and mutagenicity. Relevant to our invention are recent data indicating specifically that a lack of the GSH precursor, cysteine, rather than a decrease in biosynthetic enzyme activities is responsible for the deficiency of GSH noted in aging animals.sup.(73). Similarly, the fall in cytosolic GSH in the liver of chronic ethanol fed rats does not appear to be caused by a limitation in the capacity of gamma-glutamylcysteine synthetase activity.sup.(74).
Our studies have shown that the observed enhancement of the immune response is associated with greater production of splenic glutathione in immunized mice fed whey protein concentrate in comparison to mice fed casein, cysteine enriched casein or egg white protein in similar dietary concentration. The efficiency of dietary cysteine in inducing supernormal glutathione levels is greater when it is delivered in the whey protein than as free cysteine or within the egg white protein. Glutathione was found at higher levels in the heart and liver of whey protein fed old mice in comparison to mice fed the corresponding casein diet, the egg white protein or Purina Mouse Chow.
The use of mice as biological test subjects in research is commonly practiced world-wide. It is generally accepted that if a biological phenomenon occurs in two different mammalian species, it can be applied to other mammalian species including man. Our studies carried out in several unrelated strains of mice of both sexes therefore are of great benefit in gauging the biological activity of whey protein concentrate which appears to be independent of specific genetic or hormonal influences. Perhaps most importantly human milk has by far the highest whey protein/casein ratio than any other mammal. (See in this regard "Evolutionary Traits in Human Milk Proteins", Bounous et al, Medical Hypotheses (1988) 27, 133-140). Presumably nature has prepared humans, through the only obligatory form of nutrition, to handle undenatured whey proteins for their best metabolic advantage. In fact, one would anticipate that the favourable biological activity of whey protein concentrate in rodents might be more pronounced in the human host.